The overall goal of this protocol is to synthesize monocyte-targeting peptide amphiphile micelles for atherosclerosis imaging. This method can help answer key question in the cardiovascular field about diagnostic imaging of atherosclerotic plaque. The main advantage of this technique is that the incorporation of peptide amphiphile micelles with MCP-1 allow a greater targeting specificity and differentiation between early and late-stage atherosclerotic lesions.
The implications of this technique extend to the diagnosis of plaque at different stages of atherosclerosis, as patients with rupture-prone, late-stage plaques contain higher number of monocytes. Though this method can provide insight into atherosclerosis, it can also be applied to other diseases, such as cancer. Demonstrating the procedure with me will be Jonathan Wang, a graduate student from our laboratory.
Begin by weighing out 0.25 millimoles of fmach-L-lysine-bach-wong-resin in a reaction vessel. Transfer the vessel to a chemical fume hood and rinse the vessel sides with five milliliters of dimethylforamide. Load the reaction vessel and prepackaged amino acid vials from the C-to-N terminus, onto an automated benchtop peptide synthesizer, including an empty vial at the end for the final deprotection setp.
After running the synthesis, transfer the peptides and resin from the reaction vessel into a scintillation vial containing two to three milliliters of methanol until all of the resin has been transferred. When the resin has sunk to the bottom of the vial, remove the supernane and allow the resin to air dry, followed by vacuum drying for at least two hours at room temperature. When the vial is completely dry, use a clamp to secure the bottom half of a synthesis vessel onto an arm shaker and add two milliliters of cleavage solution to the scintillation vial.
Transfer the entire volume of peptide resin to the synthesis vessel and wash the sides of the vessel with three milliters of fresh cleavage solution. Then shake the vessel, capped, for four hours at 300 oscillations per minute. At the end of the shaking incubation, drain the cleaved peptide solution into a balanced-weighed 50 milliliter conical tube and rinse the synthesis vessel several times with the remaining cleavage solution.
Evaporate the cleaved peptide solution with nitrogen until there is less than four milliliters of solution remaining in the centrifuge tube and add 36 milliliters of ice cold ether to the peptide cleaved solution. Vortex the resulting solution. When the suspension turns completely white or yellow, pellet the peptides by centrifugation.
Resuspend the peptide in 40 milliliters of ice cold ether and vortex and sonicate the peptide again. After a second centrifugation and supernatant decanting, dry the crude peptide amphiphile pellet with nitrogen gas purging. When the ether has been visibly evaporated, add 20 milliliters of double distilled water to the tube and vortex and sonicate the tube until the peptide is completely dissolved in solution.
Freeze the solution and lifelize until dry. Then weigh the tube again and dissolve the crude peptide in five milligrams per milliliter water. Next, inject the entire volume of crude MCP-1 peptide into a high-performance liquid chromotography or HPLC instrument.
Analyze each fraction using matrix-assisted laser desorption ionization mass spectroscopy for the expected product mass charge at 2, 890. Then combine the product fractions. Blow off the acetonitrile and freeze and lifelize the purified peptides until dry.
To prepare the MCP-1 peptide amphiphiles or PAs, first dissolve the lifelized peptide in 15 milligrams per milliliter of water and combine the peptide solution with 15 milligrams per milliliter of DSPE PEG 2000 millimide solution. Add one mole of sodium hydroxide dropwise until the pH of the solution equals seven. Then nitrogen purge for five minutes, followed by overnight stirring.
The next morning, freeze and lifelize the solution. When the crude product is dry, dissolve the solid in five milligrams per milliliter water and purify the peptide by HPLC, as just demonstrated. To assemble the PA membranes, dissolve 1.75 milligrams of the MCP-1 PAs in three milliliters of methanol in a one dram vial and sonicate until the MCP-1 PA is completely dissolved.
Evaporate the methanol under nitrogen while moving the vial a circular motion and rinsing the remaining methanol off of the sides of the vial, until a uniform film is formed at the bottom. Vacuum dry the film overnight. The next morning, hydrate the film with three milliliters of water to make a 100 micromolar MCP-1 PAM solution and gently vortex and sonicate the solution until clear.
Incubate the solution at 80 degrees Celsius for 30 minutes, then cool the resulting dispersion. In the following representative experiment, MCP-1 and scrambled peptide were purified by reverse-phase HPLC on a CA column at 50 degrees Celsius using 0.1%trifluoroacetic acid in acetonitrile water mixtures. And characterized by matrix-assisted laser desorption ionization mass spectrometry, as just demonstrated.
Desisting containing peptides were conjugated onto DSPE PEG 2000 milliamide to yield DSPE PEG 2000 peptide conjugates via a thioether linkage and purified by HPLC using a C4 column. Dynamic light scattering analysis and transmission electron microscopy imaging of the monocyte targeting PAMs revealed well dispersed spherical nanoparticles with slightly positive zeta potentials. Circular dicroisms spectroscopy analysis confirmed the incorporation of the MCP-1 peptide within the micelle enhanced the secondary structure.
The incubation amalse monocytes with up to 100 micromolar concentrations of MCP-1 peptide, MCP-1 PAM, scrambled peptide, and scrambled PAM for 72 hours demonstrated the viability and biocompatibility of the peptides. The specific binding of the monocytes to MCP-1 PAMs compared to scrambled PAMs is then established by confocal microscopy. After watching this video you should have a good understanding of how to prepare monocyte targeting peptide amphiphile micelle through the synthesis and purification of the MCP-1 peptide and the preparation of the MCP-1 peptide amphiphile and micelle assembly.
Don't forget that working with chemical reagent can be extremely hazardous and that precautions, such as reading the MSDS and working inside a chemical fume hood should always be taken when working with this materials.
